The use of electrospray ionization-mass spectrometry (ESI-MS) has increased for characterizing biomolecules, for example proteins and oligonucleotides as well as other high molecular weight compounds. Collisional induced dissociation methods have provided information on amino acid and nucleotide sequence and sites of damage or modification. ESI-MS has been more recently used for analysis of non-covalent complexes including oligonucleotide duplexes, quadruplexes, and DNA-protein complexes, and probing the higher order structure of proteins. However, biological samples prepared under physiological conditions typically contain significant concentrations of salts (e.g., sodium chloride) as well as other non-volatile reagents added to stabilize samples or to maintain their enzymatic activity. The presence of small to moderate amounts of sodium salts, for example, affects electrospray stability due to changes in solution conductivity, and may significantly reduce the detected analyte ion abundance due to both suppression of ionization and the formation of multiple species having sodium adducts. Low signal-to-noise ratios of the mass spectra and poor reproducibility due to excessive adduction can result in inaccurate mass assignments and, in severe cases, even preclude spectrum interpretation.
Sodium adduct formation is attributed primarily to electrostatic interactions of sodium ions with negatively charged sites on the high molecular weight molecules, e.g. phosphate groups on the polynucleotide backbone in DNA. Large DNA molecules have a proportionally higher affinity for sodium ion because of the extended polynucleotide backbone. Therefore, removal of sodium ion from large DNA molecules to the levels required to produce high quality spectra is more difficult than for small oligonucleotides.
Various methods have been used to reduce cation adduction and other types of problematic contamination due to low MW species. A common approach is the multiple buffer exchange method using a membrane cartridges. The salt-containing sample is repeatedly diluted with ammonium acetate buffer and concentrated by centrifugation, resulting in a decreased salt concentration. This process, although simple in nature, typically requires several hours and often results in significant sample losses, which greatly limits its analytical capability for limited sample sizes. The desalting efficiency of this technique is very low for DNA samples due to the high affinity of DNA molecules for sodium, and significant cation adduct ions are still present even after more than five cycles of buffer exchange.
Although on-line LC/MS can tolerate small to moderate amounts of salt, and desalting columns are commercially available, they are not suitable for many ESI-MS experiments where samples may precipitate on the column, interact with the solvent, or when very high salt concentrations are present. It is less effective for desalting of large oligonucleotides.
Stults and Marsters, Rapid Commun. Mass Spectrom. 1991, 5, 359-363, reported an ammonium acetate precipitation method that reduced the sodium adduction for oligonucleotides up to 77-mer in length thereby improving the mass spectrum quality. This method was performed off-line, separately from the ESI-MS, required large amount of sample to overcome significant sample loss during the precipitation step. The ammonium acetate method has been shown to be less effective for desalting of larger (&gt;60-mer in size) DNA or RNA molecules.
Nordhoffet al., Rapid Commun. Mass Spectrom. 1992, 6, 771-776, described another off-line method of sodium removal by using cation exchange polymer beads, which was limited to oligonucleotide samples with low concentrations of cation contamination.
Emmett and Capioli, J. Am. Soc. Mass Spectrom. 1994, 5, 605-613, reported an on-line micro-LC (liquid chromatography) sample clean-up method using a C18 cartridge stationary phase that suffered from the disadvantages of HPLC desalting and the difficulties of packing the capillary columns.
Greig and Griffey, Rapid Commun. Mass Sepctrom. 1995, 9, 97-102, used organic bases, 25 mM piperidine and 25 mM imidazole, to suppress adduct formation or cation adduction in ESI-MS of oligonucleotide samples. But, the salt concentration tolerance of this method is relatively low (&lt;&lt;10 mM salt or cation). Further compensating for high salt concentration by addition of higher concentrations of organic bases (&gt;50 mM) resulted in greatly decreased detection sensitivity. In addition, some modified DNA and RNA molecules (e.g. DNA/drug or RNA/drug adduct) are not stable under these basic conditions.
Cheng et al., Anal. Chem. 1995, 67, 586-593, used inorganic acids and bases. Addition of organic or inorganic acids (or bases) is useful for smaller oligonucleotides, but not for larger oligonucleotides since they can alter the native conformation of DNA molecules which is undesirable for studies requiring a native DNA conformation. These methods of salt removal are useful only for salt concentrations less than about 25 mM.
Dialysis has been used for clean-up of biopolymers for removal of low molecular weight contaminants including salts. However, conventional "batch" dialysis methods require large amount of samples, proceed for several hours and incur significant sample loss. When only a small amount of sample is available, sample loss can become even more pronounced. If non-covalent association are of interest, slow desalting in conventional dialysis is more likely to result in denaturation and/or complex dissociation in solution.
Thus, there remains a need for an apparatus and method for removing contaminants from biomolecular samples that preserves the biomolecules and operates in a short time.